The goal of this program is to advance current DNA nanoparticle (NP) delivery and expression technologies to develop safe and effective therapies for important ocular disorders affecting the photoreceptor (PR) and retinal pigment epithelial (RPE) cells. The program will merge experts with molecular bioengineering, vision science, physics, and chemistry to accelerate essential steps for the generation of effective ocular non-viral gene therapy. The DNA NPs consist of single molecules of DNA compacted with lysine-PEG polycations and have a minimum diameter of 8-11 nm. Their small size, coupled with a specific uptake mechanism that efficiently traffics the NPs to the nucleus and bypasses the lysosomal degradation system, likely accounts for the ability of the NPs to robustly transfect post-mitotic, differentiated cells. The counter-ion used at the time of formulation determines the NP shape and both rod-like and ellipsoidal NPs show robust transfection of ocular cells, including RPE, PRs and retinal ganglion cells (RGCs). Moreover, murine RDS NPs have demonstrated partial phenotypic correction in a retinitis pigmentosa mouse model of RDS haploinsufficiency. Having previously shown proof-of-principle for the effective use of these NPs in the eye, in this application we will take the necessary steps to optimize the particles for clinical ocular use. First, we will optimize the NP formulation (NP shape, size, and chemical composition) for PR- and RPE-specific gene transfer (Aim #1). Second, we will engineer clinically-appropriate DNA vectors (Aim #2) to generate persistent, high levels of transgene expression. Thirds, we will test the ability of these NPs to target the macula in a non-human primate model (baboon) (Aim #3) including an assessment of whether non-invasive intravitreal delivery of NPs can transfect macular/foveal cones. This is a novel and critical development for clinical application of this technology, as many retinal degenerations target the macula. Furthermore, it is a step that cannot be modeled by a mouse system due to the absence of a macula in the rodent retina. We will also conduct toxicology and DNA biodistribution studies in baboons, including a detailed evaluation of brain visual pathways. Building on the documented ability of the current NP formulation to penetrate deep retinal layers, we hypothesize that this efficiency can be improved by the NP formulation optimization program detailed in Aim #1, enabling robust foveal cone gene transfer. In summary, results from this application will facilitate preclinical trial evaluations of DNA NPs for ocular gene delivery. PUBLIC HEALTH RELEVANCE: This program is designed to advance our compacted DNA nanotechnology to facilitate its future use as a clinical gene therapy treatment for ocular diseases. Preclinical studies are planned to lay the foundation for use of this technology as a clinically viable gene delivery system for ocular diseases such as retinitis pigmentosa (RP), various macular dystrophies (MD), and diabetic retinopathy. The minimum diameter (8-11 nm) of our NPs is much smaller than any viral vector, and may permit uniquely effective and safe intravitreal ocular dosing to deliver genes into RPE cells and foveal cones.